Enzymes that harness the extreme reactivity of electron-deficient free radical species carry out some of the most difficult chemical reactions in biology. The regio- and stereo-selectivity achieved by these enzymes defies long-held ideas that radical reactions are non-specific. This class includes the following: ribonucleotide reductases, which catalyze the first unique step in DNA biosynthesis, prostaglandin H-synthase, the target of aspirin and other non- steroidal anti-inflammatory drugs, the S-adenosylmethionine-dependent enzymes, with over six-hundred members that catalyze a wide range of radical- mediated redox reactions, and the coenzyme B12 (adenosylcobalamin)-dependent enzyme superfamily, whose members catalyze metabolite covalent bond rearrangements. The common primary step in these chemically-disparate catalyses is metal-assisted generation of an electron-deficient organic radical. This initiator radical, either by itself or through secondary radical species, promotes hydrogen atom abstraction from the substrate to form a substrate- based radical, opening a new reaction channel that facilitates transformation to the product. An outstanding issue is how the radical pair is stabilized against rapid recombination to achieve productive reaction in high yield. Elucidating the basic principles of how protein and cofactor guide radical generation, stabilization, intra-protein radical migration, and radical rearrangement will be sustained focuses of the proposed studies. The coenzyme B12-dependent enzymes, and ethanolamine ammonia-lyase specifically, have been selected for scrutiny. The contributions of molecular structure and dynamics to enzyme function will be studied by using techniques of pulsed-electron paramagnetic resonance and visible/near-infrared absorption spectroscopy, in cryotrapped and time-resolved systems on time scales ranging from picoseconds to hours. The fundamental insights and novel methods developed will promote identification and characterization of radical intermediates in other biological reactions, inform the design of programmed radical reactivity, and assist molecular-therapeutic efforts to combat pernicious free radical processes. PUBLIC HEALTH RELEVANCE: Enzymes that harness the extreme reactivity of electron-deficient free radicals perform some of the most difficult reactions in biology. We seek to understand the contributions of molecular structure and dynamics to this productive radical reactivity in coenzyme B12-dependent enzymes by using spectroscopic techniques. The fundamental insights and novel approaches developed will promote characterization of radical intermediates in other biological systems, inform the design of programmed radical reactivity, and assist molecular- therapeutic efforts to combat pernicious free radical processes.